The present invention relates generally to the discovery of co-factors for trophic factors which stimulates and/or potentiates the activity and/or specificity of trophic factors, methods of use thereof.
Trophic factors have a broad range of biological activities and their activity and specificity may be achieved by cooperation with other factors. Although trophic factors are generally active at extremely low concentrations, high concentrations of mitogen together with high cell density are often required to induce proliferation of multipotent neural stem cell populations. Growth factors for early progenitors may be useful for enhancing the success of gene transfer into stem cells as well as treating disorders by renewal of mature cells from the stem cell pool. In vitro assays using recombinant neurotrophic factors have indicated that members of a neurotrophic gene family may play sequential and complementary roles during development of the adult nervous system.
Neurotropic factors that have been identified include NT-4, NT-5, NT-6, NT-7, CNTF (ciliary neuronotrophic factor ), GDNF (Glial cell line-derived neurotrophic factor), and Purpurin. NSE (neuron-specific enolase) has been found to be a neuronal survival factor. Other factors possessing a broader spectrum of functions and which have neurotrophic activities, but are not normally classified as neurotrophins, also exist. These factors include EGF (epithelial growth factor), HBNF (heparin-binding neurite-promoting factor), IGF-2, a-FGF and b-FGF , PDGF, NSE (neuron-specific enolase), and Activin A. Other factors have been identified which specifically influence neuronal differentiation and influence transmitter phenotypes without affecting neuronal survival. Although the intracerebral administration of FGF-2 has been shown to stimulate neurogenesis in the adult rat SVZ, FGF-2 alone in the adult rat hippocampus has a limited effect on the proliferation of neural stem/progenitor cells (Kuhn et al. (1997); Wagner et al. (1999) each herein incorporated by reference).
Mitogenic growth factors, like fibroblast growth factor-2 (FGF-2) (Gage, F. H., et al., 1995, Proc. Natl Acad. Sci. USA 92:11879-11883) and epidermal growth factor (EGF) (Lois, C., and Alvarez-Buylla, A., 1993, Proc. Natl. Acad. Sci. USA 90(5):2074-2077), induce proliferation of neural progenitor cells isolated from the brain. Studies from single cells in culture demonstrate that FGF-2 (Gritti, A., et al., 1996, J. Neurosci. 16:1091-1100) and EGF (Reynolds, B. A., and Weiss, S., 1996, Develop. Biol. 175:1-13) are mitogens for multipotent neural stem cells and likely cooperate with trophic factors (Cattaneo, E., and McKay, R., 1990, Nature 347:762-765; Stemple, D. L., and Anderson, D. J., 1992, Cell 71:973-985), some of which are yet unknown (Davis, A. A., and Temple, S., 1994, Nature 372:263-266; Temple, S., 1989, Nature 340:471-473; Kilpatrick, T. J., and Bartlett, P. F., 1993, Neuron 10:255-265; Palmer, T. D., et al., 1997, Mol. Cell. Neurosci. 8:389-404) to achieve specificity. There is in vitro and in vivo evidence for the existence of stem cells in the embryonic and adult brain (Altman, et al., J. Comp. Neurol. 124:319-335, 1965; Ericksson, et al., Nature Medicine, 1998, 40:1313-1317).
The trophic factors are of potential clinical interest since they influence the functional activities and survival of distinct neural populations within the peripheral and central nervous system. Many of these molecules are currently under investigation as therapeutic agents for the treatment of neurodegenerative disorders and nerve injury, either individually or in combination with other trophic factors.
Disorders of the central nervous system encompass numerous afflictions such as neurodegenerative diseases (e.g., Alzheimer""s and Parkinson""s), acute brain injury (e.g., stroke, head injury). In recent years, neurodegenerative disease has become an important concern due to the expanding elderly population which is at greatest risk for neurodegenerative disorders, such as Alzheimer""s Disease, Multiple Sclerosis, Huntington""s Disease, and Parkinson""s Disease.
The present invention is based on the discovery and isolation of a co-factor for trophic factors. It has been discovered that trophic factors require a co-factor to stimulate and/or potentiate the trophic factor activity and/or specificity. This was clearly identified in low density cells where trophic factors are unable, or at best, at minimal levels, able to proliferate undifferentiated cells without a co-factor. In a particular embodiment of the present invention, there is provided a composition comprising glycosylated cystatin C, (CCg), an FGF co-factor that stimulates proliferation of neural and fibroblast associated undifferentiated cells. The N-glycosylation of cystatin C is required for its activity. Moreover, CCg acts in cooperation with basic fibroblast growth factor (FGF-2) to induce neural progenitor cell proliferation.
In a first preferred embodiment, substantially purified glycosylated cystatin C (CCg) polypeptide, and biologically active fragments thereof, are provided.
In another embodiment, a method for stimulating and/or potentiating trophic factor activity and/or specificity is provided. In a preferred embodiment, a method for inducing proliferation of a mammalian neural stem or progenitor cell is provided. This method includes contacting a neural stem or progenitor cell invention compositions, or a biologically active fragment thereof, alone or in combination with trophic factors such as fibroblast growth factor-2 (FGF-2), or a biologically active fragment thereof, under conditions that allow proliferation of the cell.
A method is also provided for inhibiting trophic factor activity and/or specificity, comprising administering inactive forms of invention polypeptides. Preferably, the present invention inhibits proliferation of a mammalian neural cell. The method includes contacting the cell with a non-glycosylated form of cystatin C (CCg) or an inhibitory peptide thereof (or of the CCg) under conditions that inhibit proliferation of the cell. In one aspect, the method further includes administering an FGF-2 inhibitory agent, such as an antibody, in addition to a non-glycosylated CCg or other inhibitory fragment of CCg.
In another embodiment, an assay system is provided for identifying co-factors for trophic factors, comprising purifying from conditioned medium a co-factor for trophic factors. The method comprises contacting cells cultured in a medium at low density, and thereafter purifying co-factors which stimulate and/or potentiate the trophic factor activity and/or specificity.
A method of identifying an agent which promotes neural cell proliferation is also provided. The method comprises contacting a neural undifferentiated cell with a test agent and with a trophic factor, such as fibroblast growth factor-2, under conditions that allow the components to interact. The ability of the neural undifferentiated cell to proliferate in the presence of the agent is compared with the ability of a neural undifferentiated cell, to proliferate in the absence of the agent.
In yet another embodiment, a method is provided for modulating FGF mediated processed comprising administering a co-factor for FGF to a subject.
In another embodiment, a method is provided for ameliorating a neural disorder in a subject by administering a therapeutically effective amount of glycosylated cystatin C to the subject.
A pharmaceutical composition is provided which contains a therapeutically effective amount of glycosylated cystatin C and a pharmaceutically acceptable carrier. In another embodiment, the composition includes FGF-2.
An antibody which binds glycosylated cystatin C with a different affinity than non-glycosylated cystatin C is further provided.
In yet another embodiment, substantially purified stem cell glycosylated cystatin C polypeptide, is provided by a method comprising purifying glycosylated cystatin C glycoprotein is also provided. The method includes, producing conditioned media from a neural progenitor or stem cell; performing affinity chromatography on the media; eluting a fraction containing mitogenic activity; performing papain chromatography on the fraction having mitogenic activity; and recovering a glycoprotein of about 21 kDa.